Becker Björn, Schmitt Manfred J
Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University;
Molecular and Cell Biology, Department of Biosciences and Center of Human and Molecular Biology (ZHMB), Saarland University.
J Vis Exp. 2017 Dec 15(130):56588. doi: 10.3791/56588.
Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy. Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation. Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.
细菌和植物A/B毒素利用真核细胞中的天然运输途径到达其位于细胞质中的细胞内靶标并最终导致细胞死亡。此类A/B毒素通常由具有酶活性的A亚基（例如，蓖麻毒素A（RTA））和一个或多个细胞结合B亚基组成，这些B亚基负责毒素与特定细胞表面受体的结合。我们目前对A/B毒素如何有效毒害细胞的了解，有助于科学家理解高等真核细胞中的基本细胞机制，如内吞作用和细胞内蛋白质分选。从医学角度来看，识别主要的毒素运输途径对于为患者找到合适的治疗方案或最终开发基于毒素的癌症治疗应用同样重要。由于对哺乳动物细胞中A/B毒素运输进行全基因组分析复杂、耗时且昂贵，因此已经在酵母模式生物酿酒酵母中进行了多项关于A/B毒素运输的研究。尽管酵母的细胞过程没那么复杂，但酵母和高等真核细胞中的基本细胞过程是相似的，而且在酵母中获得的结果通常可以推广到哺乳动物的情况。在此，我们描述了一种快速且易于使用的报告基因检测方法，用于分析酵母中RTA的细胞内运输。新检测方法的一个重要优点是，不仅有机会研究RTA从内质网（ER）逆向转运到细胞质中，还能研究从质膜到内质网的内吞作用和逆向毒素运输。该检测方法利用了一种报告质粒，通过体内翻译后绿色荧光蛋白（GFP）的荧光发射间接测量RTA毒性。由于RTA通过28S rRNA脱嘌呤有效地阻止蛋白质生物合成的起始，该检测方法可以通过检测荧光发射的变化来鉴定参与细胞内RTA运输的宿主细胞蛋白。
